Krisp: A Python package to aid in the design of CRISPR and amplification-based diagnostic assays from whole genome sequencing data.

Recent pandemics like COVID-19 highlighted the importance of rapidly developing diagnostics to detect evolving pathogens. CRISPR-Cas technology has recently been used to develop diagnostic assays for sequence-specific recognition of DNA or RNA. These assays have similar sensitivity to the gold standard qPCR but can be deployed as easy to use and inexpensive test strips. However, the discovery of diagnostic regions of a genome flanked by conserved regions where primers can be designed requires extensive bioinformatic analyses of genome sequences. We developed the Python package krisp to aid in the discovery of primers and diagnostic sequences that differentiate groups of samples from each other, using either unaligned genome sequences or a variant call format (VCF) file as input. Krisp has been optimized to handle large datasets by using efficient algorithms that run in near linear time, use minimal RAM, and leverage parallel processing when available. The validity of krisp results has been demonstrated in the laboratory with the successful design of a CRISPR diagnostic assay to distinguish the sudden oak death pathogen Phytophthora ramorum from closely related Phytophthora species. Krisp is released open source under a permissive license with all the documentation needed to quickly design CRISPR-Cas diagnostic assays.

Open Access and Reproducibility in Plant Pathology Research: Guidelines and Best Practices.

The landscape of scientific publishing is experiencing a transformative shift toward open access, a paradigm that mandates the availability of research outputs such as data, code, materials, and publications. Open access provides increased reproducibility and allows for reuse of these resources. This article provides guidance for best publishing practices of scientific research, data, and associated resources, including code, in The American Phytopathological Society journals. Key areas such as diagnostic assays, experimental design, data sharing, and code deposition are explored in detail. This guidance aligns with that observed by other leading journals. We hope the information assembled in this paper will raise awareness of best practices and enable greater appraisal of the true effects of biological phenomena in plant pathology.

Comparative Genomic Analysis of 31 Phytophthora Genomes Reveals Genome Plasticity and Horizontal Gene Transfer.

Phytophthora species are oomycete plant pathogens that cause great economic and ecological impacts. The Phytophthora genus includes over 180 known species, infecting a wide range of plant hosts, including crops, trees, and ornamentals. We sequenced the genomes of 31 individual Phytophthora species and 24 individual transcriptomes to study genetic relationships across the genus. De novo genome assemblies revealed variation in genome sizes, numbers of predicted genes, and in repetitive element content across the Phytophthora genus. A genus-wide comparison evaluated orthologous groups of genes. Predicted effector gene counts varied across Phytophthora species by effector family, genome size, and plant host range. Predicted numbers of apoplastic effectors increased as the host range of Phytophthora species increased. Predicted numbers of cytoplasmic effectors also increased with host range but leveled off or decreased in Phytophthora species that have enormous host ranges. With extensive sequencing across the Phytophthora genus, we now have the genomic resources to evaluate horizontal gene transfer events across the oomycetes. Using a machine-learning approach to identify horizontally transferred genes with bacterial or fungal origin, we identified 44 candidates over 36 Phytophthora species genomes. Phylogenetic reconstruction indicates that the transfers of most of these 44 candidates happened in parallel to major advances in the evolution of the oomycetes and Phytophthora spp. We conclude that the 31 genomes presented here are essential for investigating genus-wide genomic associations in genus Phytophthora. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Openness and Computational Reproducibility in Plant Pathology: Where We Stand and a Way Forward.

Open research practices have been highlighted extensively during the last 10 years in many fields of scientific study as essential standards needed to promote transparency and reproducibility of scientific results. Scientific claims can only be evaluated based on how protocols, materials, equipment, and methods were described; data were collected and prepared; and analyses were conducted. Openly sharing protocols, data, and computational code is central to current scholarly dissemination and communication, but in many fields, including plant pathology, adoption of these practices has been slow. We randomly selected 450 articles published from 2012 to 2021 across 21 journals representative of the plant pathology discipline and assigned them scores reflecting their openness and computational reproducibility. We found that most of the articles did not follow protocols for open science and failed to share data or code in a reproducible way. We propose that use of open-source tools facilitates computationally reproducible work and analyses, benefitting not just readers but the authors as well. Finally, we provide ideas and suggest tools to promote open, reproducible computational research practices among plant pathologists. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Whole Genome Sequencing-Based Tracing of a 2022 Introduction and Outbreak of Xanthomonas hortorum pv. pelargonii.

Globalization has made agricultural commodities more accessible, available, and affordable. However, their global movement increases the potential for invasion by pathogens and necessitates development and implementation of sensitive, rapid, and scalable surveillance methods. Here, we used 35 strains, isolated by multiple diagnostic laboratories, as a case study for using whole genome sequence data in a plant disease diagnostic setting. Twenty-seven of the strains were isolated in 2022 and identified as Xanthomonas hortorum pv. pelargonii. Eighteen of these strains originated from material sold by a plant breeding company that had notified clients following a release of infected geranium cuttings. Analyses of whole genome sequences revealed epidemiological links among the 27 strains from different growers that confirmed a common source of the outbreak and uncovered likely secondary spread events within facilities that housed plants originating from different plant breeding companies. Whole genome sequencing data were also analyzed to reveal how preparatory and analytical methods can impact conclusions on outbreaks of clonal pathogenic strains. The results demonstrate the potential power of using whole genome sequencing among a network of diagnostic labs and highlight how sharing such data can help shorten response times to mitigate outbreaks more expediently and precisely than standard methods.

Genetic Diversity, Evolution, and Diagnosis of Sugarcane Yellow Leaf Virus from 19 Sugarcane-Producing Locations Worldwide.

Sugarcane yellow leaf virus (SCYLV), the causal agent of yellow leaf, has been reported in an increasing number of sugarcane-growing locations since its first report in the 1990s in Brazil, Florida, and Hawaii. In this study, the genetic diversity of SCYLV was investigated using the genome coding sequence (5,561 to 5,612 nt) of 109 virus isolates from 19 geographical locations, including 65 new isolates from 16 geographical regions worldwide. These isolates were distributed in three major phylogenetic lineages (BRA, CUB, and REU), except for one isolate from Guatemala. Twenty-two recombination events were identified among the 109 isolates of SCYLV, thus confirming that recombination was a significant driving force in the genetic diversity and evolution of this virus. No temporal signal was found in the genomic sequence dataset, most likely because of the short temporal window of the 109 SCYLV isolates (1998 to 2020). Among 27 primers reported in the literature for the detection of the virus by RT-PCR, none matched 100% with all 109 SCYLV sequences, suggesting that the use of some primer pairs may not result in the detection of all virus isolates. Primers YLS111/YLS462, which were the first primer pair used by numerous research organizations to detect the virus by RT-PCR, failed to detect isolates belonging to the CUB lineage. In contrast, primer pair ScYLVf1/ScYLVr1 efficiently detected isolates of all three lineages. Continuous pursuit of knowledge of SCYLV genetic variability is therefore critical for effective diagnosis of yellow leaf, especially in virus-infected and mainly asymptomatic sugarcane plants.

Risk of Epidemic Development in Nurseries from Soil Inoculum of Phytophthora ramorum.

Soilborne inoculum arising from buried, infested leaf debris may contribute to the persistence of Phytophthora ramorum at recurrently positive nurseries. To initiate new epidemics, inoculum must not only survive but also produce sporangia during times conducive to infection at the soil surface. To assess this risk, we performed two year-long experiments in a soil plot at the National Ornamentals Research Site at Dominican University of California. Inoculated rhododendron leaf disks were buried at a depth of 5 or 15 cm in the early summer of 2014 or 2015. Inoculum was baited at the soil surface with noninfested leaf disks (2014 only) and then retrieved to assess pathogen viability and sporulation capacity every 5 weeks. Two 14-week-long trials were conducted in 2016. We were able to consistently culture P. ramorum over all time periods. Soil incubation rapidly reduced the capacity of inoculum to sporulate, especially at 5 cm; however, sporulation capacity increased with the onset of seasonally cooler temperatures. P. ramorum was baited most frequently between November and January, especially from inoculum buried at 5 cm 1 day before the baiting period; in January we also baited P. ramorum from inoculum buried at 15 cm the previous June. We validate prior observations that P. ramorum poses a greater risk after exposure to cooler temperatures and provide evidence that infested leaf debris plays a role in the perpetuation of P. ramorum in nurseries. This work provides novel insights into the survival and epidemic behavior of P. ramorum in nursery soils.

Repeated Emergence of Sudden Oak Death in Oregon: Chronology, Impact, and Management.

It has been two decades since the first detection of the sudden oak death pathogen Phytophthora ramorum in Oregon forests. Although the epidemic was managed since its first discovery in 2001, at least three invasions of three separate variants (clonal lineages), NA1, EU1, and NA2, are documented to have occurred to date. Control of this epidemic has cost over US$32 million from 2001 to 2020. This is dwarfed by the predicted cost of the closure to the Coos Bay export terminal, estimated at $58 million per year, if the epidemic was allowed to spread unchecked. Management efforts in Oregon have reduced inoculum and limited the spread of the pathogen. An outreach and citizen scientist program has been piloted to help in early detection efforts and search for disease-resistant tanoak. This feature article documents the repeated emergence, impact, costs, and lessons learned from managing this devastating invasive pathogen.

Is Disease Induced by Flooding Representative of Nursery Conditions in Rhododendrons Infected with Phytophthora cinnamomi or P. plurivora?

The degree of flooding commonly used to induce disease in Phytophthora root rot studies rarely occurs in container nurseries. Instead, over-irrigation and poor drainage result in plants periodically sitting in shallow pools of water. Rhododendron plants were grown in a noninfested substrate or substrate infested with Phytophthora cinnamomi or P. plurivora to determine whether root rot induced by flooding represents disease that occurs under simulated nursery conditions when plants are in a shallow pool of water (saucers), or are allowed to freely drain and maintained at ∼75% container capacity (CC). Generally P. cinnamomi caused more disease than P. plurivora, and all water treatments were conducive to root rot. In experiment 1, the amount of disease caused by flooding was similar to that in the saucer treatment (75% CC not tested) while in experiment 2, flooding often caused more rapid and severe disease than the saucer or 75% CC treatment. Pathogens differed in their response to water treatments. P. cinnamomi caused more disease in treatments with >90% substrate moisture for either a short (flood) or long duration (saucer), while P. plurivora was less capable of causing disease when soil moisture was maintained >90% than when substrate moisture was maintained at a more moderate level (flood, 75% CC). Our results indicate that it is not necessary to flood plants to induce disease under experimental conditions and that disease induced by flooding can represent disease in container nurseries when containers are in pools of water or maintained at ∼75% CC. In addition, our results suggest that P. cinnamomi is a more aggressive pathogen than P. plurivora in nursery conditions where drainage is poor; however, both species are capable of causing a similar amount of disease under more typical irrigation management.

First report of the NA2 clonal lineage of the sudden oak death pathogen, Phytophthora ramorum, infecting tanoak in Oregon forests.

Phytophthora ramorum Werres, de Cock & Man in't Veld, causal agent of sudden oak death (SOD) and ramorum leaf blight, is comprised of four clonal lineages in its invasive ranges of North America and Europe (Grünwald et al. 2012, Van Poucke et al. 2012). Of these, three - the NA1, NA2, and EU1 lineages - are found in U.S. nurseries, but only two, the NA1 and EU1 lineages, have been found infecting trees in North American forests (Grünwald et al. 2012, 2016). In the spring of 2021, tanoak (Notholithocarpus densiflorus Manos, Cannon & Oh) displaying symptoms consistent with SOD were detected north of Port Orford (Curry County, Oregon). Symptoms were canopy dieback and blackened petiole and stem lesions on tanoak sprouts. The pathogen isolated on PAR (CMA plus 200 ml/L ampicillin, 10 mg/L rifamycin, 66.7 mg/L PCNB) selective media was determined to be P. ramorum based on characteristic morphology of hyphae, sporangia, and chlamydospores (Werres et al. 2001). Positive identification as P. ramorum was obtained with a lineage-specific LAMP assay targeting an NA2 orphan gene, indicating the presence of the NA2 lineage. NA2 was confirmed by sequencing a portion of the cellulose binding elicitor lectin (CBEL) gene using CBEL5U and CBEL6L primers (Gagnon et al. 2014). Sequences (GenBank accessions MZ733981 and MZ733982) were aligned against reference sequences for all lineages (Gagnon et al. 2014) confirming the presence of NA2. Lineage determination as NA2 was further confirmed at eleven SSR loci (ILVOPrMS145, PrMS39, PrMS9C3, ILVOPrMS79, KI18, KI64, PrMS45, PrMS6, ILVOPrMS131, KI82ab, and PrMS43) using the methods of Kamvar et al. (2015). We completed Koch's postulates using potted tanoaks, wound-inoculated at the midpoint of 1-year old stems with either hyphal plugs or non-colonized agar (n=4 per treatment). Tanoaks were maintained in a growth chamber (20°C-day / 18°C-night temperatures) with regular watering and an 18-photoperiod using F32T8 fluorescent bulbs (Phillips, Eindhoven, The Netherlands). After 7 days, brown to black lesions 1.2 to 2.9 cm in length were observed on the inoculated stems, from which P. ramorum was subsequently re-isolated; no symptoms were observed on the controls, and no pathogens were recovered when plating the wound sites in PAR. This is the first detection of the NA2 lineage causing disease in forests worldwide. The outbreak was found on private and public lands in forests typical to the SOD outbreak in Oregon (mixed conifer and tanoak), and was 33 km north of the closest known P. ramorum infestation. Follow-up ground surveys on adjacent lands have identified over 100 P. ramorum-positive tanoak trees, from which additional NA2 isolates have been recovered from bole cankers. NA2 is thought to be more aggressive than the NA1 lineage (Elliott et al. 2011), which has been present in Curry County since the mid-1990s (Goheen et al. 2017). Eradication of the NA2 lineage is being pursued to slow its further spread and prevent overlap with existing NA1 and EU1 populations. The repeated introductions of novel lineages into the western United States native plant communities highlights the vulnerability of this region to Phytophthora establishment, justifying continued monitoring for P. ramorum in nurseries and forests. References • Elliott, M, et al. 2011. For. Path. 41:7. https://doi.org/10.1111/j.1439-0329.2009.00627.x • Gagnon, M.-C., et al. 2014. Can. J. Plant Pathol. 36:367. https://doi.org/10.1080/07060661.2014.924999 • Goheen, E.M., et al. 2017. For. Phytophthoras 7:45. https://doi: 10.5399/osu/fp.7.1.4030 • Grünwald, N. J., et al. 2012. Trends Microbiol. 20:131. https://doi.org/10.1016/j.tim.2011.12.006 • Grünwald, N. J., et al. 2016. Plant Dis. 100:1024. https://doi.org/10.1094/PDIS-10-15-1169-PDN • Kamvar, Z.N. et al. 2015. Phytopath. 105:982. https://doi.org/10.1094/PHYTO-12-14-0350-FI • Van Poucke, K., et al. 2012. Fungal Biol. 116:1178. https://doi.org/10.1016/j.funbio.2012.09.003 • Werres, S., et al. 2001. Mycol. Res. 105: 1155. https://doi.org/10.1016/S0953-7562(08)61986-3.

Phylogeography of the wide-host range panglobal plant pathogen Phytophthora cinnamomi.

Various hypotheses have been proposed regarding the origin of the plant pathogen Phytophthora cinnamomi. P. cinnamomi is a devastating, highly invasive soilborne pathogen associated with epidemics of agricultural, horticultural and forest plantations and native ecosystems worldwide. We conducted a phylogeographic analysis of populations of this pathogen sampled in Asia, Australia, Europe, southern and northern Africa, South America, and North America. Based on genotyping-by-sequencing, we observed the highest genotypic diversity in Taiwan and Vietnam, followed by Australia and South Africa. Mating type ratios were in equal proportions in Asia as expected for a sexual population. Simulations based on the index of association suggest a partially sexual, semi-clonal mode of reproduction for the Taiwanese and Vietnamese populations while populations outside of Asia are clonal. Ancestral area reconstruction provides new evidence supporting Taiwan as the ancestral area, given our sample, indicating that this region might be near or at the centre of origin for this pathogen as speculated previously. The Australian and South African populations appear to be a secondary centre of diversity following migration from Taiwan or Vietnam. Our work also identified two panglobal, clonal lineages PcG1-A2 and PcG2-A2 of A2 mating type found on all continents. Further surveys of natural forests across Southeast Asia are needed to definitively locate the actual centre of origin of this important plant pathogen.

Phylogeographic Approaches to Characterize the Emergence of Plant Pathogens.

Phylogeography combines geographic information with phylogenetic and population genomic approaches to infer the evolutionary history of a species or population in a geographic context. This approach has been instrumental in understanding the emergence, spread, and evolution of a range of plant pathogens. In particular, phylogeography can address questions about where a pathogen originated, whether it is native or introduced, and when and how often introductions occurred. We review the theory, methods, and approaches underpinning phylogeographic inference and highlight applications providing novel insights into the emergence and spread of select pathogens. We hope that this review will be useful in assessing the power, pitfalls, and opportunities presented by various phylogeographic approaches.

Novel Introductions and Epidemic Dynamics of the Sudden Oak Death Pathogen Phytophthora ramorum in Oregon Forests.

Sudden oak death caused by Phytophthora ramorum has been actively managed in Oregon since the early 2000s. To date, this epidemic has been driven mostly by the NA1 clonal lineage of P. ramorum, but an outbreak of the EU1 lineage has recently emerged. Here, we contrast the population dynamics of the NA1 outbreak first reported in 2001 to the outbreak of the EU1 lineage first detected in 2015. We performed tests to determine whether any of the lineages were introduced more than once. Infested regions of the forest were sampled between 2013 and 2018 (n = 903), and strains were genotyped at 15 microsatellite loci. Most genotypes observed were transient, with 272 of 358 unique genotypes emerging during one year and disappearing the next year. The diversity of EU1 was very low and isolates were spatially clustered (less than 8 km apart), suggesting a single EU1 introduction. Some forest isolates are genetically similar to isolates collected from a local nursery in 2012, suggesting the introduction of EU1 from this nursery or simultaneous introduction to both the nursery and latently into the forest. In contrast, the older NA1 populations were more polymorphic and spread more than 30 km2. A principal component analysis supported two to four independent NA1 introductions. The NA1 and EU1 epidemics infest the same area but show disparate demographics because of the initial introductions of the lineages spaced 10 years apart. Comparing these epidemics provides novel insight regarding patterns of emergence of clonal pathogens in forest ecosystems.

Transcriptome-Derived Amplicon Sequencing Markers Elucidate the U.S. Podosphaera macularis Population Structure Across Feral and Commercial Plantings of Humulus lupulus.

Obligately biotrophic plant pathogens pose challenges in population genetic studies due to their genomic complexities and elaborate culturing requirements with limited biomass. Hop powdery mildew (Podosphaera macularis) is an obligately biotrophic ascomycete that threatens sustainable hop production. P. macularis populations of the Pacific Northwest (PNW) United States differ from those of the Midwest and Northeastern United States, lacking one of two mating types needed for sexual recombination and harboring two strains that are differentially aggressive on the cultivar Cascade and able to overcome the Humulus lupulus R-gene R6 (V6), respectively. To develop a high-throughput marker platform for tracking the flow of genotypes across the United States and internationally, we used an existing transcriptome of diverse P. macularis isolates to design a multiplex of 54 amplicon sequencing markers, validated across a panel of 391 U.S. samples and 123 international samples. The results suggest that P. macularis from U.S. commercial hop yards form one population closely related to P. macularis of the United Kingdom, while P. macularis from U.S. feral hop locations grouped with P. macularis of Eastern Europe. Included in this multiplex was a marker that successfully tracked V6-virulence in 65 of 66 samples with a confirmed V6-phenotype. A new qPCR assay for high-throughput genotyping of P. macularis mating type generated the highest resolution distribution map of P. macularis mating type to date. Together, these genotyping strategies enable the high-throughput and inexpensive tracking of pathogen spread among geographical regions from single-colony samples and provide a roadmap to develop markers for other obligate biotrophs.

Molecular Phylogenomics and Population Structure of Phytophthora pluvialis.

Phytophthora pluvialis is an oomycete that was first isolated from soil, water, and tree foliage in mixed Douglas-fir-tanoak forests of the U.S. Pacific Northwest (PNW). It was then identified as the causal agent of red needle cast of radiata pine (Pinus radiata) in New Zealand (NZ). Genotyping-by-sequencing was used to obtain 1,543 single nucleotide polymorphisms across 145 P. pluvialis isolates to characterize the population structure in the PNW and NZ. We tested the hypothesis that P. pluvialis was introduced to NZ from the PNW using genetic distance measurements and population structure analyses among locations between countries. The low genetic distance, population heterozygosity, and lack of geographic structure in NZ suggest a single colonization event from the United States followed by clonal expansion in NZ. The PNW Coast Range was proposed as a presumptive center of origin of the currently known distribution of P. pluvialis based on its geographic range and position as the central cluster in a minimum spanning network. The Coastal cluster of isolates were located at the root of every U.S. cluster and emerged earlier than all NZ clusters. The Coastal cluster had the highest degree of heterozygosity (Hs = 0.254) and median pairwise genetic distance (0.093) relative to any other cluster. Finally, the rapid host diversification between closely related isolates of P. pluvialis in NZ indicate that this pathogen has the potential to infect a broader range of hosts than is currently recognized.

Phytophthora Species Differ in Response to Phosphorous Acid and Mefenoxam for the Management of Phytophthora Root Rot in Rhododendron.

Phytophthora root rot, caused by many soilborne Phytophthora spp., is a significant disease affecting the $42 million rhododendron nursery industry. Rhododendron growers have increasingly reported failure by two systemic fungicides, phosphorous acid and mefenoxam, to adequately control root rot. Both fungicides may be applied as a foliar spray or soil drench but it is unknown how application method, fungicide chemistry, or pathogen diversity affects disease control. Therefore, two experiments were conducted to (i) determine whether differences in application method or fungicide chemistry affect control of root rot caused by P. cinnamomi and P. plurivora and (ii) evaluate the sensitivity of Phytophthora spp. and isolates from the rhododendron industry to each fungicide. Results demonstrated that soil drenches of either fungicide were more effective than foliar sprays for control of P. cinnamomi but were ineffective for P. plurivora. Furthermore, Phytophthora spp. and isolates varied in sensitivity to phosphorous acid and mefenoxam, and there were multiple fungicide-insensitive isolates, especially within P. plurivora. Differences in sensitivity were also observed among isolates from different nurseries and production systems, with some nurseries having less sensitive isolates than others and with container systems generally having less sensitive isolates than field systems. Our results provide three potential reasons for why fungicide control of Phytophthora root rot might fail: (i) the fungicide can be applied to the wrong portion of the plant for optimal control, (ii) there are differences in fungicide sensitivity among soilborne Phytophthora spp. and isolates infecting rhododendron, and (iii) fungicide-insensitive isolates are present in the rhododendron nursery industry.

Development of a Diagnostic Assay for Race Differentiation of Podosphaera macularis.

Hop powdery mildew (caused by Podosphaera macularis) was confirmed in the Pacific Northwest in 1996. Before 2012, the most common race of P. macularis was able to infect plants that possessed powdery mildew resistance based on the R-genes Rb, R3, and R5. After 2012, two additional races of P. macularis were discovered that can overcome the resistance gene R6 and the partial resistance found in the cultivar Cascade. These three races now occur throughout the region, which can complicate management and research efforts because of uncertainty on which race(s) may be present in the region and able to infect susceptible hop genotypes. Current methods for determining the races of P. macularis are labor intensive, costly, and typically require more than 14 days to obtain results. We sought to develop a molecular assay to differentiate races of the fungus possessing virulence on plants with R6, referred to as V6-virulent, from other races. The transcriptomes of 46 isolates of P. macularis were sequenced to identify loci and variants unique to V6 isolates. Fourteen primer pairs were designed for 10 candidate loci that contained single nucleotide polymorphisms (SNP) and short insertion-deletion polymorphisms. Two differentially labeled locked nucleic acid probes were designed for a contig that contained a conserved SNP associated with V6-virulence. The resulting duplexed real-time PCR assay was validated against 46 V6 and 54 non-V6 P. macularis isolates collected from the United States and Europe. The assay had perfect discrimination of V6-virulence among isolates of P. macularis originating from the western U.S. but failed to predict V6-virulence in three isolates collected from Europe. The specificity of the assay was tested with different species of powdery mildew fungi and other microorganisms associated with hop. Weak nonspecific amplification occurred with powdery mildew fungi collected from Vitis vinifera, Fragaria sp., and Zinnia sp.; however, nonspecification amplification is not a concern when differentiating pathogen race from colonies on hop. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.

First reports of Phytophthora ramorum clonal lineages NA1 and EU1 causing Sudden Oak Death on tanoaks in Del Norte County, California.

A year of forest health surveys has led to the first detection of Phytophthora ramorum in Del Norte County followed by the first wildland detection of the EU1 clonal lineage (Grunwald et al. 2009) of this pathogen in California. In July 2019, leaves were sampled from two tanoaks (Notholithocarpus densiflorus) and 16 California bay laurels (Umbellularia californica) in Jedediah Smith State Park in Del Norte County, the northernmost coastal County of California. Leaves displayed lesions normally associated with Sudden Oak Death (SOD) caused by P. ramorum and were discovered during the citizen science-based survey known as SOD Blitz (Meentemeyer et al. 2015). Samples were surface sterilized using 75% Ethanol and plated on PARPH-V8 agar (Jeffers and Martin 1986). After plating, DNA was extracted and amplified using two P. ramorum-specific assays (Hayden et al. 2006, Kroon et al. 2004). Leaves from two tanoaks exhibiting twig die-back had typical SOD lesions along the midvein, gave positive PCR results and yielded cultures with colony morphology, sporangia and chlamydospores typical of the NA1 lineage of P. ramorum originally isolated in California from tanoaks and coast live oaks (Quercus agrifolia) (Rizzo et al. 2002). The ITS locus and a portion of the Cox-1 locus were sequenced from DNA extracts of each culture using primers DC6-ITS4 (Bonants et al. 2004) and COXF4N-COXR4N (Kroon et al. 2004), respectively. ITS sequences (GB MN540639-40) were typical of P. ramorum and Cox-1 sequences (GB MN540142-3) perfectly matched the Cox-1 sequence of the NA1 lineage (GB DQ832718) (Kroon et al. 2004). Microsatellite alleles were generated as described in Croucher et al. (2013) for the two Del Norte cultures and for eight P. ramorum cultures, representative of the four main multilocus genotypes (MLGs) present in California, namely c1 (Santa Cruz/Commercial Nurseries), c3 (San Francisco Bay Area), c2 (Monterey County), and c4 (Humboldt County) (Croucher et al. 2013). The two Del Norte MLGs were identical to one another and most similar to MLG c1, with a single repeat difference at a single locus. SSR results suggest the inoculum source may not be from Humboldt County, neighboring to the South, but from a yet unidentified outbreak, possibly associated with ornamental plants. Jedediah Smith State Park was surveyed for 12 months following the initial detection, however the pathogen has yet to be re-isolated in that location. In July 2020, SOD symptomatic leaves from two tanoak trees exhibiting twig cankers were collected 8 Km north of Jedediah Smith State Park, where three additional tanoak trees displayed rapidly browned dead canopies consistent with late stage SOD. Leaves were processed as above. Colonies from these samples produced chlamydospores and sporangia typical of P. ramorum on PARPH-V8 agar, but displayed a growth rate faster than that of NA1 genotypes and were characterized by aerial hyphae, overall resembling the morphology of EU1 lineage colonies (Brasier 2003). The EU1 lineage was confirmed by the perfect match of the sequence of a portion of the Cox-1 gene (GB MW349116-7) with the Cox-1 sequence of EU1 genotypes (GB EU124926). The EU1 clonal lineage has been previously isolated from tanoaks in Oregon forests, approximately 55 Km to the North (Grünwald et al. 2016), but this is the first report for California wildlands and will require containment and government regulations. It is unknown whether the EU1 strains in Del Norte County originated from Oregon forests or elsewhere.

Fungal Evolution in Anthropogenic Environments: Botrytis cinerea Populations Infecting Small Fruit Hosts in the Pacific Northwest Rapidly Adapt to Human-Induced Selection Pressures.

Many fungal pathogens have short generation times, large population sizes, and mixed reproductive systems, providing high potential to adapt to heterogeneous environments of agroecosystems. Such adaptation complicates disease management and threatens food production. A better understanding of pathogen population biology in such environments is important to reveal key aspects of adaptive divergence processes to allow improved disease management. Here, we studied how evolutionary forces shape population structure of Botrytis cinerea, the causal agent of gray mold, in the Pacific Northwest agroecosystems. Populations of B. cinerea from adjacent fields of small fruit hosts were characterized by combining neutral markers (microsatellites) with markers that directly respond to human-induced selection pressures (fungicide resistance). Populations were diverse, without evidence for recombination and association of pathogen genotype with host. Populations were highly localized with limited migration even among adjacent fields within a farm. A fungicide resistance marker revealed strong selection on population structure due to fungicide use. We found no association of resistance allele with genetic background, suggesting de novo development of fungicide resistance and frequent extinction/recolonization events by different genotypes rather than the spread of resistance alleles among fields via migration of a dominant genotype. Overall our results showed that in agroecosystems, B. cinerea populations respond strongly to selection by fungicide use with greater effect on population structure compared to adaptation to host plant species. This knowledge will be used to improve disease management by developing strategies that limit pathogen local adaptation to fungicides and other human-induced selection pressures present in Pacific Northwest agroecosystems and elsewhere.IMPORTANCE Agroecosystems represent an efficient model for studying fungal adaptation and evolution in anthropogenic environments. In this work, we studied what evolutionary forces shape populations of one of the most important fungal plant pathogens, B. cinerea, in small fruit agroecosystems of the Pacific Northwest. We hypothesized that host, geographic, and anthropogenic factors of agroecosystems structure B. cinerea populations. By combining neutral markers with markers that directly respond to human-induced selection pressures, we show that pathogen populations are highly localized and that selection pressure caused by fungicide use can have a greater effect on population structure than adaptation to host. Our results give a better understanding of population biology and evolution of this important plant pathogen in heterogeneous environments but also provide a practical framework for the development of efficient management strategies by limiting pathogen adaptation to fungicides and other human-induced selection pressures present in agroecosystems of the Pacific Northwest and elsewhere.

Population Diversity and Structure of Podosphaera macularis in the Pacific Northwestern United States and Other Populations.

Powdery mildew, caused by Podosphaera macularis, is one of the most important diseases of hop. The disease was first reported in the Pacific Northwestern United States, the primary hop-growing region in this country, in the mid-1990s. More recently, the disease has reemerged in newly planted hopyards of the eastern United States, as hop production has expanded to meet demands of local craft brewers. The spread of strains virulent on previously resistant cultivars, the paucity of available fungicides, and the potential introduction of the MAT1-2 mating type to the western United States, all threaten sustainability of hop production. We sequenced the transcriptome of 104 isolates of P. macularis collected throughout the western United States, eastern United States, and Europe to quantify genetic diversity of pathogen populations and elucidate the possible origins of pathogen populations in the western United States. Discriminant analysis of principal components grouped isolates within three to five geographic populations, dependent on stringency of grouping criteria. Isolates from the western United States were phenotyped and categorized into one of three pathogenic races based on disease symptoms generated on differential cultivars. Western U.S. populations were clonal, irrespective of pathogenic race, and grouped with isolates originating from Europe. Isolates originating from wild hop plants in the eastern United States were genetically differentiated from all other populations, whereas isolates from cultivated hop plants in the eastern United States mostly grouped with isolates originating from the west, consistent with origins from nursery sources. Mating types of isolates originating from cultivated western and eastern U.S. hop plants were entirely MAT1-1. In contrast, a 1:1 ratio of MAT1-1 and MAT1-2 was observed with isolates sampled from wild plants or Europe. Within the western United States a set of highly differentiated loci were identified in P. macularis isolates associated with virulence to the powdery mildew R-gene R6. The weight of genetic and phenotypic evidence suggests a European origin of the P. macularis populations in the western United States, followed by spread of the pathogen from the western United States to re-emergent production regions in the eastern United States. Furthermore, R6 compatibility appears to have been selected from an extant isolate within the western United States. Greater emphasis on sanitation measures during propagation and quarantine policies should be considered to limit further spread of novel genotypes of the pathogen, both between and within production areas.